Circadian (daily) rhythms are a crucial component of human health that regulates sleep, alertness, hormones, metabolism, and many other biological processes. The ultimate explanation for the mechanism of circadian oscillators will require characterizing the structures, functions, and interactions of the molecular components of these clocks. The current project is to elucidate the basic principles of circadian clocks at a biophysical/molecular level in a model system, the prokaryotic cyanobacteria, where genetic/biochemical studies have identified three key clock proteins, KaiA, KaiB, and KaiC. These three proteins can reconstitute a circadian oscillator in vitro; this remarkable demonstration has led to a re-evaluation of our understanding of circadian clocks in all organisms, including mammals. Moreover, the crystal structures of the KaiA, KaiB, and KaiC proteins have been reported-these are the first clock proteins to have their 3-D structures determined. The advent of atomic resolution structures of the molecular components of this circadian pacemaker marks a dramatic watershed in circadian research by ushering in truly molecular analyses of circadian mechanisms. The current project will determine the molecular basis of the core clockwork by biochemical/biophysical, genetic, and structural approaches. Three critically important unanswered questions in chronobiology are to explain how the biochemical mechanism (i) can be temperature compensated, (ii) operates rhythmic outputs under some conditions but not others, and (iii) is able to keep time accurately in the face of changes in metabolism. This project will face these issues head-on. Temperature compensation of this biological clock will be investigated by screening for temperature dependent mutants of KaiC, KaiB, and KaiA in vivo. These mutations will be mapped onto the 3-D structures of the proteins to generate specific hypotheses that will be tested by novel in vitro biochemical analyses and targeted mutations. The rate constants and other biochemical data that result from the analyses of these mutants will be integrated with our previous data to generate models that account for the temperature-compensated, 24 h time constant of the in vitro oscillator. Differential expression of circadian rhythms under some conditions but not others (conditionality) is based on novel mechanisms of codon usage in cyanobacteria, and the mechanism and adaptiveness of this fascinating phenomenon will be analyzed as well as recruited to maximize cost-effective production of biopharmaceuticals. Finally, a novel hypothesis with far-reaching implications will be analyzed, namely that accurate circadian timekeeping requires compensation for metabolic perturbations, of which temperature change is only one among many such perturbations. The answers to these questions will lead to wide-ranging insights into the mechanisms and applications of biological timekeeping.